Organic complex of platinum, its preparation and its use for treating malignant tumors

ABSTRACT

An organic complex of platinum, cis-isocitrato (1,2 diaminocyclohexane) platinum (II) having improved anti-tumor activity and water-solubility. The complex is prepared by reacting cis-dichloro (1,2-diaminocyclohexane) platinum (II) with silver nitrate, removing the silver chloride formed, and reacting the resulting liquid with trisodium isocitrate. The complex is administered in the form of a pharmaceutical composition.

This invention relates to an organic complex of platinum, a process forpreparing it and its therapeutic use for the treatment of malignanttumours.

According to one aspect of the invention, there is provided as a novelchemotherapeutic agent having anti-tumour activity cis-isocitrato(1,2-diaminocyclohexane) platinum (II), which may be abbreviated toPHIC. This organic complex of platinum can be prepared by reacting amolecular moiety in which the platinum atom is partially chelated with1,2-diaminocyclohexane (trans-d and l isomers) represented by thegeneral formula: ##STR1## with trisodium isocitrate (dl isomer), whichhas the formula: ##STR2## which acts as a labile ligand, and thereafterrecovering the resulting platinum complex from the reaction mixture. Twobonds can be formed between molecule (1) and any pair of reactivepositions 1, 2, 3 and 4 of the molecule (2). Chelation most likelyoccurs at the 1- and 2-positions and at the 1- and 3-positions.Interaction at the 1-4 3-4, 2-3 and 2-4 positions is also possible. Amonodentate interaction between (1) and (2) is also quite possible: thefirst Pt bond being a Pt-carboxylato bond, the second being a Pt--OHbond. The invention includes all possible complexes as well as mixturesof these complexes in all proportions.

In a preferred embodiment of the invention, the organic complex ofplatinum is prepared by reacting cis-dichloro (1,2-diaminocyclohexane)platinum (II) with silver nitrate to form the corresponding dinitratoplatinum compound which is thereafter reacted with trisodium isocitrateto form the required platinum complex.

The platinum complex of the invention, i.e. PHIC, has been found to haveuseful anti-tumor activity. It is useful in the treatment of malignanttumors in warm blooded animals including humans. Apart from itsanti-tumor activity, PHIC has also been found to exhibit anti-microbialactivity and to be useful as a disinfectant.

Thus in accordance with another aspect of the invention, there isprovided a method of treating malignant tumours in a human subjectcomprising the administration to the subject of an effective dose ofPHIC.

It will be appreciated that for chemotherapeutic use, PHIC will normallybe administered in the form of a pharmaceutical composition comprisingPHIC as an active ingredient in association with a pharmaceuticallyacceptable diluent or excipient therefor. For the treatment of malignanttumours, PHIC may be administered orally, intramuscularly orintravenously. Hence the pharmaceutical composition will normally bemade up in a form appropriate to the desired mode of administration,preferably in a dosage unit form containing a discrete amount of theactive ingredient PHIC. For oral administration the dosage unit may be,for example, a capsule, pellet or packaged powder, whereas forintravenous or intramuscular administration, the dosage unit may be, forexample, a discrete quantity of an injectable solution or suspensionpackaged in a suitable sterile container, such as an ampoule.

For the treatment of malignant tumours, the daily dosage of PHIC willgenerally be from about 1 to about 400 mg per kilogram body weight ofthe treated subject. It may be administered in one or severaladministrations.

The inhibition of cellular proliferation by, and the anti-tumor activityof, platinum coordination complexes, in particular cis-dichlorodiammineplatinum (II), which may be abbreviated to PDD, of the formula: ##STR3##were discovered some fifteen years ago. Since that time, much researchhas been devoted to this category of compound in the hope of first,identifying the most active members, and second, of overcoming thedisadvantages which were rapidly found to present, these compounds asknown to in particular, their low degree of solubility which rendersadministration difficult, their nephrotoxicity and ototoxicity. Researchhas also been done to elucidate their mechanism of action. Recentstudies have shown that the solubility of these compounds, theiranti-tumor activity and their nephrotoxic effects can be favourablyinfluenced by causing various alterations into the molecule. Forinstance, the replacement of the two chlorine atoms in PDD by bidentateligands such as oxalato and malonato, was proposed as early as 1973.Then, in 1974, the replacement of the two NH₃ molecules in PDD by1,2-diaminocyclohexane was suggested. The complex so obtained had theadvantage of not causing any cross-resistance to PDD in the animaltreated.

However, these investigations were not pursued because of the low watersolubility of all these compounds. Research was thus directed to ligandspossessing a third carboxylic group which could enhance solubility ofthe complex and possibly improve its therapeutic activity withoutnecessarily increasing its toxicity.

These ligands include sodium trans-aconitate and sodium citratemolecules, the latter being present in the living human organism (Krebscycle) in both the citric and isocitric isomeric forms. Differentcorresponding complexes have been synthesised in order to compare theirproperties as will be described below. It was observed that the citratecomplex PHIC exhibited a greater solubility than compound (3).

The present invention is concerned with the behaviour and properties ofthe isocitrate complex, PHIC. In comparison with all the compoundsprepared both in previous investigations and in the course of thepresent study, this isocitrate complex presents the following unexpectedadvantages:

1. An almost unlimited degree of solubility which is at least severalhundred times greater than that of other similar platinum complexes.

2. Extremely low toxicity in mice and baboons.

3. Very marked anti-tumor activity toward L1210 leukemia and sarcoma180.

The comparative trials described below quantitate these advantages,demonstrate this outstanding combination of properties and consequentlyshow the therapeutic value of this compound in the treatment of tumours.

These comparative studies were undertaken with the following compoundswhich are identified by abbreviations for greater convenience:

I. COMPOUNDS SYNTHESISED FROM CIS-DICHLORODIAMINE PLATINUM (II) OR PDDOF FORMULA ##STR4## PAC: cis-citratodiammineplatinum (II) PAIC:cis-isocitratodiammineplatinum (II)

PATA: cis-(t-aconitato) diammineplatinum (II)

II. COMPOUNDS SYNTHESISED FROM CIS-DICHLORO(1,2-DIAMINOCYCLOHEXANE)PLATINUM (II) OR DIS-Pt (DAC) Cl₂ OR PHD ##STR5## PHC: cis-citrato(1,2-diaminocyclohexane) platinum (II) PHIC: cis-isocitrato(1,2-diaminocyclohexane) platinum (II)

PHTA: cis-(t-aconitato) (1,2-diaminocyclohexane) platinum (II)

The compound of the invention, i.e. PHIC, can be prepared from compound(4) as follows:

In the first step, the dihydrate derivative was prepared in accordancewith the following reaction:

    cis-Pt(DAC)Cl.sub.2 +2AgNO.sub.3 →cis-[Pt(DAC)(H.sub.2 O).sub.2 ](NO.sub.3).sub.2 +2AgCl

In the second step, the isocitrate anion was complexed as follows:

    cis-[Pt(DAC)(H.sub.2 O).sub.2 ](NO.sub.3).sub.2 +trisodium→PHIC, 3H.sub.2 O+NaNO.sub.3 +HNO.sub.3 isocitrate 2H.sub.2 O

The following Example illustates the preparation of PHIC.

EXAMPLE

In 250 ml of bidistilled water were suspended 5 g of cis-Pt (DAC)Cl₂ and4.3 g of silver nitrate were added. The reaction medium was protectedfrom light. The reaction itself took place at 50°-55° C. over a periodof 4 hours. A white precipitate of silver chloride formed. The reactionmedium was then brought to 0°-4° C. for several hours and then filteredon Whatman paper. (The word "Whatman" is a registered Trademark ofWhatman Inc., New Jersey, U.S.A.). The precipitate was washed with water(50-75 ml) and the resulting colorless filtrate (300-350 ml) was passedthrough a Millipore filter (0.45 microns). (The word "Millipore" is aregistered Trademark of the Millipore Corporation, Massachusetts,U.S.A.). To the filtrate, 3.85 g of trisodium isocitrate, 2H₂ O wereadded and the mixture was brought to 60°-65° C. for 20 hours duringwhich time it was shielded from light. The resulting yellow-coloredsolution (which can be filtered on Whatman paper if necessary) was thenconcentrated to 25-30 ml using a rotavapor (temperature of the bath =50°C.).

The resulting complex was filtered on a Millipore filter (0.45 microns)and added by means of a pipette to 1000 ml of 1/1 cold ethanol/acetonemixture under continuous stirring. The medium was allowed to stand at 4°C. for 2 hours, then filtered on sintered glass, washed with 100 mlacetone and then with 100 ml ether.

The off-white complex obtained was dried under vacuum and then weighed.In this manner, about 6.5 g of PHIC were collected from 5 g of cis-Pt(DAC)Cl₂, which corresponds to a yield of 85%.

The PHIC so obtained was in the form of a fine off-white powder whichbegan to turn black when heated to 245° and then completely decomposedat 254° C. The following results were obtained in the elementaryanalysis of PHIC (Pt was determined by atomic absorption):

    ______________________________________                                                     C    H      N      Pt   Cl   Na                                  ______________________________________                                        PHIC.3H.sub.2 O calculated                                                                   24.26  4.10   4.72 32.84                                                                              --   7.16                              M.W. = 594.10 found                                                                          24.10  3.96   4.76 32.10                                                                              --   6.99                              ______________________________________                                    

These analytical results are also in agreement with the formula PHIC.2H₂O in which the Pt (DAC) moiety does not form two bonds with theisocitrate ion, but is linked with one carboxylate ion, most likely C-1,the other bond being a Pt--OH bond.

This compound has an exceptionally high degree of solubility. In waterit is almost unlimited (more than 1500 mg/ml). In a 9% solution ofsodium chloride it is about 700 mg/ml, the complex changing in 24 hoursto a yellow chlorinated derivative which precipitates. PHIC can beidentified by its chromatogram and its spectrum analysis (U.V., I.R. and¹³ C) which are illustrated in the accompanying drawings.

In FIG. 1 are shown the high pressure liquid chromatograms of thefollowing compounds:

A: PDD

B, C, D and E: PHIC

F and H: trisodium isocitrate

G: PAC

obtained under the following experimental conditions:

    __________________________________________________________________________              Concentration                                                                         Volume               Flow                                             of the  Injected             rate Pressure                          Compound  Solvent (μl)                                                                            Sensitivity                                                                         Eluent    (ml/min)                                                                           (psi)                             __________________________________________________________________________    A. PDD    0.5 mg/ml                                                                             25   0.01  9% NaCl solun                                                                           1.5  1500                                        9% NaCl sol.                                                        B. PHIC   0.5 mg/ml                                                                             25   0.02  H.sub.2 O 1.5  1500                                        H.sub.2 O                                                           C. PHIC*  0.5 mg/ml                                                                             15   0.04  C.sub.2 H.sub.5 OH/H.sub.2 O                                                            24/6)                                                                              300                                         C.sub.2 H.sub.5 OH/H.sub.2 O                                                  (4/6)                                                               D. PHIC*  0.5 mg/ml                                                                             50   0.01  1% acetic acid                                                                          4    300                                         1% acetic acid                                                      E. PHIC*  0.5 mg/ml                                                                             50   0.01  1% acetic acid                                                                          2    300                                         1% acetic acid                                                      F. ISOCITRATE*                                                                          3 mg/ml 10   0.04  C.sub.2 H.sub.5 OH/H.sub.2 O                                                            24/6)                                                                              300                                         C.sub.2 H.sub.5 OH/H.sub.2 O                                                  (4/6)                                                               G. PAC    5 mg/ml 20   0.5   H.sub.2 O 1.3  1000                                        H.sub.2 O                                                           H. ISOCITRATE*                                                                          5 mg/ml 10   0.4   1% acetic acid                                                                          4    300                                         1% acetic acid                                                      __________________________________________________________________________     *column C.sub.18 radial reverse                                          

In FIG. 2 are shown the U.V. spectra of the indicated compounds namely:

trisodium citrate

PAC

PHC

trisodium isocitrate

PAIC

PHIC

obtained under the following experimental conditions:

The U.V. spectra were recorded on a CARY 14 spectrophotometer. All ofthe complexes as well as the unreacted ligands absorbed between 203 and204 nm. The concentrations and molecular absorption coefficients ε in 1.mol⁻¹.cm⁻¹) were the following:

trisodium citrate: 10 mg/100 ml H₂ O; ε=590

PAC: 2.5 mg/100 ml H₂ O; ε=7500

PHC: 2.5 mg/100 ml H₂ O; ε=10100

trisodium isocitrate: 6 mg/100 ml H₂ O; ε=620

PAIC: 3 mg/100 ml H₂ O; ε=6200

PHIC: 3 mg/100 ml H₂ O; ε=9400

In FIG. 3 is shown the I.R. spectrum of PHIC recorded on a Perkin Elmer577 spectrophotometer. The solid compound was diluted to 1% withpotassium bromide and compressed into pellets. The two standard peakscorrespond to ν₁ =2850.7 cm⁻¹ and ν₂ =906.7 cm⁻¹.

Finally, in FIG. 4 are shown the ¹³ C spectra of trisodium isocitrate,PHS and PHIC obtained under the following experimental conditions:

The ¹³ C resonance spectra with proton decoupling, were obtained using apulsed Fourrier Transform apparatus, Bruker WH 90, working at 22.62 MHz.The number of scans was 400 for isocitrate, 3000 for PHS and 17000 forPHIC, with a sweepwidth of 6 KHz and a 8K memory. In all cases a 90°pulse of 12 μs was used with a repetition rate of 3s.

The chemical shifts δ Ci are expressed in ppm and are compared to TMS.The compounds were dissolved in D₂ O at the concentration of 1 g/ml forisocitrate, 0.44 g/ml for PHS 1.1 g/ml for PHIC and the solutions werefiltered on a Millipore filter (0.45 microns) before the spectrum wasrecorded. The C₆ D₆ was used as external standard. The chemical shiftsare given in the table below. The PHS was included in the study in orderto obtain the ¹³ C spectrum of the 1,2-diaminocyclohexane ligandcomplexed with Pt (II). CHEMICAL SHIFTS δ ¹³ C (ppm/TMS) OF ISOCITRATE,PHS AND PHIC

    ______________________________________                                        Peak           Assign-                                                        No   δCi ment    Formula                                                ______________________________________                                        ISOCITRATE                                                                    123456                                                                             183.4183.1182.5 75.6 51.2 39.6                                                              C-2C-3C-1C-4C-5C-6                                                                   ##STR6##                                                                     PHS                                                  12345                                                                              69.434.633.0 31.425.5                                                                       C-1,1'C-2,2'C-3,3'                                                                   ##STR7##                                            ______________________________________                                        Peak           Assignment                                                     No    δCi    ligand       Ci                                            ______________________________________                                        PHIC                                                                          1    184.2                                                                    2    183.9                                                                    3    183.8                                                                    4    182.9                                                                    5    182.7                                                                    6    182.4         ISO*         C-1,2,3                                       7    182.3                                                                    8    182.2                                                                    9    182.0                                                                    10   181.9                                                                          75.9                                                                    11                 ISO          C-4                                                 75.2                                                                          64.8                                                                    12                 DAC*         C-1,1'                                              64.0                                                                          51.1                                                                          50.6                                                                    13    50.3         ISO          C-5                                                 48.9                                                                          39.6                                                                          39.1                                                                    14    38.4         ISO          C-6                                                 36.4                                                                          34.3                                                                          34.1                                                                    15    33.4         DAC          C-2,2'                                              32.8                                                                    16    26.3         DAC          C-3,3'                                        ______________________________________                                         *ISO = isocitrate;                                                            DAC = 1,2diaminocyclohexane                                              

The anti-tumour properties of PHIC were evaluated, in comparativestudies, using classical tests on two kinds of experimental tumours inmice, leukemia L 1210 and sarcoma S 180. The experimental conditionsused and the results obtained are described below:

I. LEUKEMIA L 1210: Ascite and Solid

DAB mice/2 females (CNRS, Orleans) of 20 to 22 g

10 mice per dose

i.p.=intraperitoneal route; i.v.=intravenous route;

s.c.=subcutaneous route

(a) i.p. graft--i.p. treatment

10⁵ cells were grafted intraperitoneally on D₀ and intraperitonealtreatment was carried out 24 hours later, D₁, in one single injection.

(b) i.p. graft--i.v. treatment

10⁵ cells were grafted intraperitoneally on D₀ and intravenous treatmentin the tail vein was carried out 24 hours later, D₁, in one singleinjection.

(c) s.c. graft--i.p. treatment

10⁶ cells were grafted under the skin behind the head on D₀ andintraperitoneal treatment was carried out in one single injection eitheron D₁ or on D₄.

II. SARCOMA S180: ASCITE

Swiss female mice (Evic-Ceba, Bordeaux) of 20 to 22 g

10 mice per dose

10⁶ cells intraperitoneally grafted on D₀

intraperitoneal treatment 24 hours later, D₁, in one single injection.

The compounds investigated, namely PHIC, PDD, PAC, PAIC, PHC and PHTA,were dissolved in a 9% solution of sodium chloride. Two other compounds,namely the cis-sulfatomonoaquo(1,2 diaminocyclohexane) platinum (II),abbreviated PHS, and the cis-malonato (1,2 diaminocyclohexane) platinum(II), abbreviated PHM, have already been reported and will be includedin this study for comparison purposes. PHD and PHM were suspended in a4% Klucel (hydroxy propyl cellulose) solution (the word "Klucel" is aregistered trademark of Hercules Incorporated, Delaware, U.S.A.).

PHS was dissolved in water because its dissolution in 9% aqueous sodiumchloride gave rise almost instantaneously to the formation of a yellowinsoluble chlorinated compound, most likely monochlorinated thendichlorinated PHD. The solutions of these compounds were preparedextemporaneously. The increase in life span, ILS (%), were calculated incomparison with the controls by the following formula: ##EQU1## Deathswere recorded until day 30 for L 1210 tumour and until day 60 for S180tumour. Survivals were included in the ILS calculation by assigning a 30day life span in the L 1210 experiments and a 60 day life span in the S180 experiments It should be added that most of the mice reaching 30days of survival treatment with PHM and PHIC developed peritonitis asPHM and PHIC developed peritonitis as judged by adhesion and hardeningof the abdominal viscera. This effect was also observed in healthy micewhen the compound was injected by intraperitoneal route. In contrast,this delayed toxic effect was never encountered when the compound wasadministered by intravenous route. This type of toxicity seems to becorrelated with the presence of 1,2-diaminocyclohexane (PHD, PHS, PHM,PHC and PHIC) since it was never observed with the diammino derivatives(PPD, PAC, PAIC).

The compounds PAC, PAIC and PHTA gave 17% (100 mg/kg), 74% (75 mg/kg)and 13% (500 mg/kg) of ILS in leukemia L 1210 (10⁵ cells,intraperitoneal graft on D₀, treatment in one single injection on D₁).It is important to note the following:

(a) PHIC exerts the anti-tumour activity in leukemia L 1210 and sarcomaS 180, intraperitoneal graft, but causes peritonitis intraperitonealtreatment (Tables I and II).

(b) PHIC is active against leukemia L 1210 when the graft isintraperitoneal and the treatment is intravenous (Table III).

(c) The therapeutic index of PHIC, defined by the ratio T.I.=LD₅₀ /MED(MED=Minimum effective dose, i.e., the dose corresponding to an ILS of25% for both tumours) in 5.5 times greater than that of PDD in the caseof leukemia L 1210 and 4.0 times greater in the case of sarcoma S 180.

                  TABLE I                                                         ______________________________________                                        COMPARED ANTI-TUMOUR ACTIVITY OF PDD AND PHIC                                 AGAINST LEUKAEMIA L 1210 AND SARCOMA S 180                                               L 1210.sup.a                                                                              S 180.sup.b                                                    Dose              Survivals     Survivals                             Compound                                                                              (mg/kg)  ILS (%)  (%)    ILS (%)                                                                              (%)                                   ______________________________________                                        PDD     2        28       --     --                                                   4        52       0      41     0                                             6        75       0      --     --                                            8        106      0      65     0                                     PHIC    5        16       0      --     --                                            10       40       0      29     0                                             25       59       0      46     0                                             50       79       0      112    20                                            100.sup.c                                                                              129      20     171    40                                    ______________________________________                                         .sup.a I.p. graft (10.sup.5 cells) on D.sub.0 and i.p. treatment on           D.sub.1, 10 mice per dose average survival time for control animals: PDD      9.3 ± 0.8 days; PHIC 8.6 ± 0.4 days                                     .sup.b I.p. graft (10.sup.6 cells) on D.sub.0 and i.p. treatment on           D.sub.1, 10 mice per dose average survival time for control animals: PDD      17.1 ± 2.0 days; PHIC = 18.7 ± 1.3 days                                 .sup.c one toxic death excluded from ILS calculation                     

                  TABLE II                                                        ______________________________________                                        COMPARED ANTI-TUMOUR ACTIVITY AGAINST                                         LEUKAEMIA L 1210 AND SARCOMA S 18O                                                       L 1210      S 180                                                          Dose              Survivals     Survivals                             Compound                                                                              (mg/kg)  ILS (%)  (%)    ILS (%)                                                                              (%)                                   ______________________________________                                        PHD     1        5        0      100    0                                             2        49       0      128    20                                            4        65       0      127    10                                    PHM     5        --       --      25    0                                             10       --       --      36    0                                             20       --       --      44    0                                             40       --       --      60    0                                             50       103      10      96    0                                     PHS     0.5      --       --      4     0                                             1        --       --      10    0                                             2        --       --      25    0                                             4        --       --      47    0                                             5        112      40     112    10                                    PHC     10       --       --      20    0                                             25       --       --     124    20                                            50       --       --     121    30                                            100      --       --     --     --                                            120       90*     0      --     --                                    ______________________________________                                         *with 5 mice only                                                             I.p. graft (10.sup.5 cells for L 1210 tumour and 10.sup.6 cells for S 180     tumour) on D.sub.O and i.p. treatment on D.sub.1, 10 mice per dose       

                  TABLE III                                                       ______________________________________                                        ANTI-TUMOUR ACTIVITY OF PHIC AGAINST                                          LEUKAEMIA L 1210 S.C. GRAFT - I.P. TREATMENT                                  I.P. GRAFT - I.V. TREATMENT                                                   ______________________________________                                                      Subcutaneous Graft (10.sup.6 cells).sup.a                       I.p. treatment on D.sub.1                                                                   ILS                                                             62.5 mg/kg    26                                                              I.p. treatment on D.sub.4                                                     62.5 mg/kg    17                                                              125 mg/kg     50                                                                            Intraperitoneal Graft (10.sup.5 cells).sup.b                    I.v. treatment on D.sub.1                                                                   ILS                                                             50 mg/kg      25                                                              125 mg/kg     41                                                              ______________________________________                                         .sup.a average survival time for control animals = 8.6 ± 0.5 days          .sup.b average survival time for control animals = 8.8 ± 0.7 days     

Comparative studies were also performed in order to evaluate the invitro and in vivo toxicities of the compound PHIC of the invention. Theresults of the trials are summarized:

A. L 1210 CELLS IN CULTURE

In vitro toxicity was determined using L 1210 cells in culture bymeasuring the inhibitory dose 50, ID₅₀, which represents theconcentration of the platinum compound which reduces by 50% the rate ofgrowth of the cells after prolonged contact, usually 24 to 48 hours,with the compound to be tested. At the ID₅₀, all the cells were viableas judged by exclusion with trypan blue. The experimental part of thisstudy is described in a recently published article (P. LECOINTE, J. P.MACQUET and J. L. BUTOUR (1979) Biochem. Biophys. Res. Commun., 90,209-213). The results are shown in Table IV.

                  TABLE IV                                                        ______________________________________                                        CYTOTOXICITY OF THE DIFFERENT COMPOUNDS ON                                    L 1210 CELLS IN CULTURE                                                       Compounds compared                                                                              ID.sub.50 (μM)                                           ______________________________________                                        PDD               2.33                                                        PHD               0.13                                                        PHS               0.19                                                        PHM               0.39                                                        PHIC              0.42                                                        ______________________________________                                    

B. TOXICITY IN THE MOUSE AND IN THE RAT.

1. Toxicity i.p., i.v. and p.o.

Acute toxicity, one single injection of the compound, was measured usingSwiss female mice (Evic-Ceba, Bordeaux) of 21 to 23 g. andSprague-Dawley rats. Ten animals were treated for each dose studied. Thetoxicity of the different compounds is given in Table V.

                  TABLE V                                                         ______________________________________                                        ACUTE TOXICITY OF THE DIFFERENT COMPOUNDS IN                                  THE MOUSE                                                                     PDD       PAC    PAIC    PHD  PHC  PHIC   PHTA                                ______________________________________                                        LD.sub.0 *                                                                          9       125    75    7    15O  153    >500                              (mg/kg)                              (LD.sub.10)*                             ______________________________________                                         LD.sub.0 * = maximum nonlethal dose;                                          LD.sub.10 = dose killing 10% of the animals.                             

The toxicity by intraperitoneal (i.p., 10 animals per dose), intravenous(i.v., 10 animals per dose) and oral (p.o., 10 animals per dose) routesof PHIC is reported in Table VI. The loss of weight on the 4th day ofthe animals i.p. treated was 3.5 g of the LD₁₀ dose, 4.3 g for the LD₅₀dose.

                  TABLE VI                                                        ______________________________________                                        TOXICITY BY INTRAPERITONEAL (I.P.), INTRAVENOUS                               (I.V.) AND ORAL (P.O.) ROUTES OF PHIC IN THE                                  MOUSE AND RAT                                                                                 LD.sub.10  LD.sub.50                                                                              LD.sub.90                                 animal   route  mg/Kg      mg/Kg    mg/Kg                                     ______________________________________                                        mouse    I.P.   135        236      365                                                                  (220-254)*                                                  I.V.   348        421      489                                                                  (411-431)*                                                  P.O.              >1 000                                             rat      I.P.    61        143      278                                                                  (134-153)*                                                  I.V.   111        173      244                                                                  (156-192)*                                                  P.O.              >1 000                                             ______________________________________                                         LD.sub.10 = dose killing 10% of the animals                                   LD.sub.50 = dose killing 50% of the animals                                   LD.sub.90 = dose killing 90% of the animals                                   *range corresponding to P = 0.05                                         

It should be added that between 50 and 80% of the surviving animals atthe i.p. treatment developed peritonitis as judged by adhesion andhardening of the abdominal viscera within the month following theinjection of PHIC. As discussed above this problem seems to be linked tothe presence of the 1,2-diaminocyclohexane ligand. When PHIC is injectedintravenously this long-term toxic effect is not observed.

2. Renal and hepatic toxicity

The nephrotoxicity of PDD is well known. It was thus of interest tocompare the effects of the novel compound of the invention with those ofthe already known compounds listed above on the kidney and the liver,i.e. the organs in which the greatest amount of platinum accumulates.

Swiss female mice (Evic-Ceba, Bordeaux) of 20 to 22 g were treated byintraperitoneal route D₀ and then sacrificed on D₁, D₄ and D₁₀. Ninemice were used per compound and the doses administered are given inTable VII.

                  TABLE VII                                                       ______________________________________                                        DOSES USED IN THE STUDY OF RENAL AND HEPATIC                                  TOXICITY                                                                      Com-                                                                          pound  PDD     PAC    PHD  PHM   PHS  PHC  PHIC                               ______________________________________                                        Dose   8       100    4    50    5    120  150                                (mg/kg)                                                                              .BHorizBrace.                                                                            .BHorizBrace.                                               Solvent                                                                              9.permill. aqueous                                                                       4.permill. aqueous                                                                       H.sub.2 O                                                                          H.sub.2 O                                                                          9.permill.                                    solution of                                                                              Klucel solu-         aqueous                                       NaCl       tion                 solution                                                                      of NaCl                                ______________________________________                                    

It should be noted that these doses corresponded to the maximumnon-toxic doses in the healthy mouse (˜LD₀). The renal and hepaticparenchyma were observed in organs fixed with a Duboscq-Brasil mixtureand embedded in paraffin. Slices 5 microns thick were coloured withhemalun eosine stain. In most cases, the alterations were located in thecortex and in the proximal and distal tubules. They were:

(a) Nuclear alterations:

marked anisocaryosis

alteration of the chromatin either washed out or lumpy withhyperchromatism and/or pyknosis

the nuclear membrane was irregular and sometimes incised

(b) Alterations of the cytoplasm:

Anisocytosis with alteration of the brush border of the proximal tubulesand intracytoplasmic eosinophilic inclusions.

Some scattered patches suggested isolated cellular necrosis in theproximal and distal tubules. In one case an abscess was observed on D₁₀after treatment with PHM. Tubular dilation with hyaline casts in thenephron as a whole were found with, at times, a pseudovesicular aspect.These alterations, when they were observed, were at their maximum on D₄and stabilized on D₁₀. Variations in the same series suggestedvariations in sensitivity.

These alterations were observed with:

(a) PDD (8 mg/kg), PAC (100 mg/kg) and PHM (50 mg/kg).

(b) PHD (4 mg/kg): vascular alterations without any hyaline casts.

(c) PHS (5 mg/kg): vascular dilation and proximal distorted tubules.

PHC (120/mg/kg) and PHIC (150 mg/kg) did not provoke any significantalterations on this type of preparation. The splenic and even suprarenalparenchyma which were sometimes dissected, showed no abnormalities.

From the foregoing it can be concluded that the novel compound of theinvention, PHIC, does not cause any renal lesion even at a dost 20 timesgreater to that of PDD.

Comparative studies of the behavious of the compound of the invention,PHIC and of compound PDD have also been made with respect to cellularpenetration in vivo in the mouse. The results are given below. DBA/2mice received 10⁶ L 1210 cells on D₀ and were treated three days laterwith an amount of platinum compound equal to the LD₀ in the healthymouse. Two hours after treatment on D₃, the animals were sacrificed.About 20.10⁶ cells were extracted from the peritoneum and washed severaltimes so as to eliminate the extra-cellular platinum and also anyintracellular platinum which was not bound in a covalent manner with thecellular organelles. Platinum was monitored by atomic absorption forwhole cells and also on the DNA which was purified by phenol extraction.In the case of PDD (9 mg/kg) only 0.4% of the platinum injected into theanimal entered the cells. This corresponds to 180.10⁻¹⁶ g of platinumper leukemic cell. Under these conditions, one fixed platinum atom per3000 nucleotides was found, i.e. a total of 1% of the amount whichpenetrated the cells was complexed to DNA. For animals treated with 150mg/kg PHIC, 0.15% penetrated the cells, which corresponds to 700. 10⁻¹⁶of platinum per cell. For this compound one fixed platinum atom wasfound for 3500 nucleotides. As in the case of PDD, 1% of the amount ofPHIC which entered the cells was complexed to DNA. The results are givenin Table VIII.

                  TABLE VIII                                                      ______________________________________                                        COMPARISON OF THE PENETRATION OF PDD AND                                      PHIC INTO L 1210 CELLS GRAFTED TO THE                                         ANIMAL AND OUANTIFICATION IN THE DNA                                                             Amount of pene-                                                                            Number of fixed                                                  trating plati-                                                                             platinum atoms                                Platinum           num per cell per nucleotide                                compound                                                                              Dose (mg/kg)                                                                             (× 10.sup.-16 g)                                                                     on DNA                                        ______________________________________                                        PDD      9         180          1/30000                                       PHIC    150        700          1/3500                                        ______________________________________                                    

Finally, a preliminary toxicological study was conducted with PHIC inthe baboon so as to determine the folowing parameters:

(a) Nephrotoxicity

(b) Hematotoxicity

(c) Gastro-intestinal toxicity

(d) Hepatic toxicity

(e) Ototoxicity

as well as the maximum non-lethal dose.

In order to find out the maximum tolerable dose, two baboons, identifiedby no 1 and no 2, received 100 mg/kg and 150 mg/kg respectively withoutprehydration. Hydration was performed on baboon no 3 which received adose of 200 mg/kg.

    ______________________________________                                                                       Frequency                                      Baboon No                                                                             Weight (kg)                                                                              Dose (mg/kg)                                                                              of treatment                                   ______________________________________                                        1       8.4        100                  1 at D.sub.0                                                                  1 at D.sub.37                                                                 1 at D.sub.58                                                        6        1 at D.sub.84                                                                 1 at D.sub.105                                                                1 at D.sub.133                        2       7.3        150                  1 at D.sub.0                          3       7.5        200                  1 at D.sub.0                          ______________________________________                                    

The PHIC was dissolved in a 9% solution of sodium chloride at theconcentration of 100 mg/ml and then filtered on a 0.22 microns milliporefilter. The compound was injected into the femoral vein during 5 to 10minutes. Baboon No. 3 (200 mg/kg) received 700 ml of physiological serumby intravenous route. Baboons Nos. 1 and 2 received 1000 ml bysub-cutaneous route.

Gastro-intestinal toxicity was the first type of toxicity to appear andgenerally manifested itself 1 to 2 hours after injection of thecompound. In all instances, vomiting was not profuse and lasted between4 and 5 hours. Blood samples were taken at regular intervals and theresults are listed in Tables IX, X and XI. D₀ was the day of treatmentand a sample was taken before the compound was injected.

The results on D₀ were therefore control values.

D₁ =first day after treatment,

D₂ =second day, etc.

Baboon No. 2 treated with 150 mg/kg PHIC died on D₁₁, most certainlyfrom the nephrotoxicity of the compounds as indicated by a creatinelevel of 1552. This baboon had neither eaten nor drunk for 11 days.Prolonged hydration of the animal (2 to 3 days) by subcutaneous routewas also tested but appeared not to be the best solution. At 100 mg/kg,under these conditions, no nephrotoxic effect was observed, in theabsence of hydration. At a dose of 200 mg/kg with hydation,nephtotoxicity is detected but creatinine levels return to normal valueson day 18.

An ototoxicity test was carried out on baboon No. 3 (200 mg/kg) on D₃₁.This test comprised the following: (a) Registering the auditorypotentials when the ear was submitted to 1500 stimuli at 90, 80, 70, 60,50, 40, 30, 20 and 10 decibels. The results gave the latency times forconduction from the ear to the brain. (b) Performing the same kind oftest by echocochleography. This analysis was much more accurate than theprevious one because it was only carried out in the Corti organ(cochlea). Frequencies of 8000, 4000, 2000 and 1000 Hertz at 90, 70, 50,30, 20 and 10 decibels were used. Measurements were made using a RaciaMedelec Empled MK₄ 1026. No ototoxic effect was detected at the dose of200 mg/kg of PHIC. The properties of PHIC compared to those of PDD canbe summarized as follows:

    ______________________________________                                                        PHIC       PDD                                                ______________________________________                                        1.   Hydrosolubility  unlimited    2 mg/ml                                                          (>1500 mg/ml)                                           2.   Anti-tumour properties                                                        (a) Therapeutic index                                                         L 1210           36.3         6.5                                             S 180            21.5         5.2                                             (b) Surviving animals                                                                          Yes          No                                              having received                                                               L 1210 leukaemia                                                              and S 180 sarcoma                                                        3    Nephrotoxicity   No           Yes                                             (LD.sub.0 level, mice)                                                   ______________________________________                                    

The compound of the invention is superior to PDD by the criteria ofsolubility, toxicity and anti-tumor activity. (Cf. Tables IX, X and XIpp. 22, 23, 24, 25 and 26).

It will be recognized by one skilled in the art that the administrableamount depends on the seriousness of the condition to be treated andtherefore is generally administered in that amount which is effective tocontrol the tumours, which amount is in the range of 1 to 400 mg per kgof body weight per day.

For information relating to cancer and current forms of cancertreatment, the following publications are incorporated by reference:

Thorn, George W. et al., Harrisons Principals of Internal Medicine,Eighth Edition, McGraw-Hill, Inc. (New York, 1977) pp. 1731-1788).

Goodman, Louis S. et al. The Pharmacological Basis of Therapeutics,Fifth Edition, McMillan Publishing Co., Inc. (New York, 1975) pp.1248-1253.

With regard to other platinum compounds having activity against leukemiaL 1210, reference is made to NTIS document No. PB-299 427,(N-phosphonacetyl-L-aspartate) (1,2-diamino-cyclohexane) platinum (II)or Alkali Metal Salt, U.S. Ser. No. 6-058,287, filed July 7, 1979, byMeischen, Gale and Noff.

                                      TABLE IX                                    __________________________________________________________________________    ANALYSIS OF BABOON No 1: 100 mg/kg                                            __________________________________________________________________________                   D.sub.0                                                                       (Control)                                                                          D.sub.1                                                                           D.sub.2                                                                           D.sub.6                                                                           D.sub.16                                                                          D.sub.30                                                                          D.sub.37                                                                          D.sub.38                                                                          D.sub.41                                                                          D.sub.48                  __________________________________________________________________________    QUICK          100  75  80  65  95  85  85  85  100 95                        PLASMA PROTHROMBIN                                                            TIME (100%)                                                                   FIBRINOGEN I   3.20 --  2.25                                                                              2.40                                                                              1.95                                                                              1.52                                                                              1.46                                                                              1.85                                                                              2.72                                                                              2.70                      (2-4 g/l)                                                                     PROACCLELERIN V (100%)                                                                       --   --  --  100 --  --  --  --  --  --                        PROCONVERTIN VII                                                                             --   --  --  70  --  --  --  --  --  --                        + STUART FACTOR X                                                             (100%)                                                                        PROTHROMBIN II --   --  --  95  --  --  --  --  --  --                        (100%)                                                                        CREATININE     117  --  110 91  105 138 99  85  102 136                       (53-130 μmols/l)                                                           GRANULOCYTES   2550 --  --  2480                                                                              4560                                                                              3969                                                                              2989                                                                              4824                                                                              4212                                                                              3696                      (4000-7000)                                                                   PLATELETS      364000                                                                             --  --  526000                                                                            414000                                                                            414000                                                                            362000                                                                            299000                                                                            371000                                                                            708000                    (100000-250000)                                                               WEIGHT         8.4  --  --  7.7 8.4 --  8.35                                                                              --  --  7.6                       (Kg)                                                                          __________________________________________________________________________                   D.sub.57                                                                           D.sub.58                                                                          D.sub.62                                                                          D.sub.76                                                                          D.sub.79                                                                          D.sub.84                                                                          D.sub.85                                                                          D.sub.90                                                                          D.sub.100                     __________________________________________________________________________    QUICK          90   --  50  95  95  --  85  --  95                            PLASMA PROTHROMBIN                                                            TIME (100%)                                                                   FIBRINOGEN I   2.67 --  1.86                                                                              1.75                                                                              2.10                                                                              --  2.08                                                                              --  2.50                          (2-4 g/l)                                                                     PROACCELERIN V (100%)                                                                        --   --  --  --  --  --  100 --  --                            PROCONVERTIN VII                                                                             --   --  --  --  --  --  90  --  --                            + STUART FACTOR X                                                             (100%)                                                                        PROTHROMBIN II --   --  --  --  --  --  --  --  --                            (100%)                                                                        CREATININE     96   --  136 121 138 --  97  96  110                           (53-130 μmols/l)                                                           GRANULOCYTES   2498 --  3726                                                                              1071                                                                              2541                                                                              --  3599                                                                              1917                                                                              3230                          (4000-7000)                                                                   PLATELETS      329000                                                                             --  378000                                                                            375000                                                                            382000                                                                            --  549000                                                                            685000                                                                            340000                        (100000-250000)                                                               WEIGHT         --   8.45                                                                              --  --  --  7.93                                                                              --  --  --                            (Kg)                                                                          __________________________________________________________________________                   D.sub.105                                                                          D.sub.106                                                                         D.sub.111                                                                         D.sub.122                                                                         D.sub.129                                                                         D.sub.133                                                                         D.sub.134                                                                         D.sub.140                         __________________________________________________________________________    QUICK          95   75  48  100 100 --  --  100                               PLASMA PROTHROMBIN                                                            TIME (100%)                                                                   FIBRINOGEN I   2.25 2.20                                                                              2.20                                                                              2.10                                                                              2.15                                                                              --  --  2.50                              (2-4 g/l)                                                                     PROACCELERIN V --   --  100 --  --  --  --  --                                (100%)                                                                        PROCONVERTIN VII                                                                             --   --  39  --  --  --  --  --                                + STUART FACTOR X                                                             (100%)                                                                        PROTHROMBIN II --   --  70  --  --  --  --  --                                (100%)                                                                        CREATININE     60   120 102 *   90  --  126 114                               (53-130 μmols/l)                                                           GRANULOCYTES   2030 10120                                                                             1121                                                                              1232                                                                              5406                                                                              --  7245                                                                              4794                              (4000-7000)                                                                   PLATELETS      266000                                                                             231000                                                                            345000                                                                            348000                                                                            508000                                                                            --  580000                                                                            441000                            (100000-250000)                                                               WEIGHT         8.59 --  --  --  --  7.65                                                                              --  --                                (Kg)                                                                          __________________________________________________________________________     D.sub.0 = 1st injection, D.sub.37 = 2nd injection.                            D.sub.58 = 3rd injection, D.sub.84 = 4th injection.                           D.sub.105  = 5th injection, D.sub.133 = 6th injection.                        The values in brackets indicate the standards found in the human being.       *BUN = 4.5 mmol/l (normal range: 3-7 mmol/l).                            

                                      TABLE X                                     __________________________________________________________________________    ANALYSIS OF BABOON No 2: 150 mg/kg                                                           D.sub.00                                                                           D.sub.0                                                                  (control)                                                                          (control)                                                                          D.sub.1                                                                           D.sub.4                                                                           D.sub.11                                     __________________________________________________________________________    QUICK          95   90   60  85  55                                           PLASMA PROTHROMBIN                                                            TIME (100%)                                                                   FIBRINOGEN I   1.85 1.46 1.46                                                                              2.82                                                                              4.20                                         2-4 g/l)                                                                      PROACCELERIN V --   --   70  --  100                                          (100%)                                                                        PROCONVERTIN VII                                                                             --   --   62  --  55                                           + STUART FACTOR X                                                             (100%)                                                                        PROTHROMBIN II --   --   42  --  55                                           (100%)                                                                        CREATININE     125  130  96  245 1552                                         (53-130 μmols/l)                                                           GRANULOCYTES   3072 6885 9672                                                                              1988                                                                              555                                          (4000-7000)                                                                   PLATELETS      507000                                                                             293000                                                                             261000                                                                            212000                                                                            174000                                       (100000-250000)                                                               WEIGHT         --   7.3  --  --  5.5                                          (Kg)                                                                          __________________________________________________________________________     D.sub.0 = 1st injection. The values in brackets indicate the standards        found in the human being.                                                

                                      TABLE XI                                    __________________________________________________________________________    ANALYSIS OF BABOON No 3: 200 mg/kg.                                                        D.sub.0                                                                       (Control)                                                                          D.sub.4                                                                           D.sub.6                                                                           D.sub.12                                                                          D.sub.15                                                                          D.sub.18                                                                          D.sub.24                                                                          D.sub.30                                                                          D.sub.40                        __________________________________________________________________________    QUICK        95   10  30  85  90  100 100 95  95                              PLASMA                                                                        PROTHROMBIN                                                                   TIME (100%)                                                                   FIBRINOGEN I 2.07 2.70                                                                              3.00                                                                              4.80                                                                              3.85                                                                              2.60                                                                              2.00                                                                              2.25                                                                              1.50                            (2-4 g/l)                                                                     PROACCELERIN V                                                                             --   100 100 --  --  --  --  --  --                              (100%)                                                                        PROCONVERTIN VII                                                                           --   <5  25  --  --  --  --  --  --                              + STUART FACTOR X                                                             (100%)                                                                        PROTHROMBIN II                                                                             --   30  25  --  --  --  --  --  --                              (100%)                                                                        CREATININE   62   140 350 646 265 118 97  104 94                              (53-130 μmols/l)                                                           GRANULOCYTES 3800 4536                                                                              5236                                                                              7905                                                                              3550                                                                              722 4872                                                                              10752                                                                             4731                            (4000-7000)                                                                   PLATELETS    241000                                                                             70000                                                                             208000                                                                            241000                                                                            114000                                                                            312000                                                                            366000                                                                            457000                                                                            299000                          (100000-250000)                                                               WEIGHT       7.5  6.25                                                                              --  5.31                                                                              --  --  5.6 5.9 6.33                            (Kg)                                                                          __________________________________________________________________________     D.sub.0 = 1st injection. The values in brackets indicate the standards        found in the human being.                                                

I claim:
 1. Cis-isocitrato (1,2-diaminocyclohexane) platinum (II).
 2. Aprocess for forming cis-isocitrato (1,2-diaminocyclohexane) platinum(II) complex which comprises reacting a platinum atom which is partiallychelated with 1,2-diaminocyclohexane together with trisodium isocitrate(dl-isomer).
 3. A process according to claim 2 wherein cis-dichloro(1,2-diaminocyclohexane) platinum (II) is reacted with trisodiumisocitrate.
 4. A process according to claim 2, wherein cis-dichloro(1,2-diaminocyclohexane) platinum (II) is first reacted with silvernitrate to form the corresponding dinitratoplatinum compound which isthereafter reacted with trisodium isocitrate to form the requiredplatinum complex.
 5. In the process of claim 2 wherein the reaction iscarried out in an aqueous medium and the platinum complex is formedincluding, the step of adding an organic solvent, whereby the recoveryof an insoluble platinum complex is promoted.
 6. The process of claim 2which comprises carrying out the reaction at a temperature above roomtemperature.
 7. The process of claim 6, which comprises prior, to addingthe organic solvent, the step of removing part of the water from thereaction mixture.
 8. The process of claim 5 in which the platinumcomplex is recovered in a yield of at least about 85%.
 9. A process forpreparing cis-isocitrato (1,2-diaminocyclohexane) platinum (II) complexwhich comprises reacting a compound of the formula:

    cis--[Pt(1,2-diaminocyclohexane)(H.sub.2 O).sub.2 ](NO.sub.3).sub.2

with trisodium isocitrate at a temperature of from about 60° to about65° C. in water and then recovering the product formed.
 10. The processof claim 9 wherein the product is recovered by the steps comprisingconcentrating the aqueous solution, filtering and then precipitating theproduct in a mixture of ethanol and acetone.
 11. A therapeuticcomposition comprising the compound cis-isocitrato (1,2diaminocyclohexane) platinum (II) complex for use in the treatment oftumors and a pharmaceutically acceptable carrier, the amount of saidplatinum II complex in the composition being a therapeutically effectiveamount and the composition being effective to inhibit L 1210 Leukemia orsarcoma 180 cells in baboons.
 12. The therapeutic composition of claim11 wherein the composition is in a dosage unit form suitable for oral,intramuscular or intravenous administration.
 13. A pharmaceuticalcomposition for inhibiting tumors in warm-blooded animals whichcomprises an effective amount of cis-isocitrato (1,2-diaminocyclohexane)platinum (II) in a pharmaceutically acceptable vehicle, whichcis-isocitrato (1,2-diaminocyclohexane) platinum (II) is effective toinhibit tumors selected from the group consisting of leukemia L1210 orsarcoma S180 in baboons.
 14. A therapeutic composition comprising thecompound cis-isocitrato (1,2-diaminocyclohexane) platinum (II) in atherapeutically effective amount, and a pharmaceutically acceptablecarrier, said compound being effective to inhibit sarcoma S 180, saidcomposition being effective for inhibiting 1210 Leukemia or sarcoma 180in baboons.
 15. A method for use in the treatment of mammals whichcomprises administering to a mammal a therapeutically effective amountof a composition comprising cis-isocitrato (1,2-diaminocyclohexane)platinum (II) complex and pharmaceutically acceptable carrier, whichcomposition is effective to inhibit L 1210 Leukemia cells in baboons.16. The method of claim 15 wherein the administration is oral,intramuscular or intravenous.
 17. The method of claim 16 wherein thecomposition is administered in a dosage range of 1 to 400 mg/kg of bodyweight per day.
 18. A method of inhibiting tumors in a warm-bloodedanimal which comprises administering to said animal an effective amountof the composition of claim 13, which platinum compound is effective toinhibit L1210 cells the baboons.
 19. A method of inhibiting tumors in awarm-blooded animal which comprises administering to said animal aneffective amount of the composition of claim 13, which composition iseffective to inhibit tumors selected from the group consisting ofleukemia L1210 or sarcoma S180 in baboons.
 20. A method for thetreatment of mammals which comprises administering to a mammal aneffective amount of a composition which comprises cis-isocitrato(1,2-diaminocyclohexane) platinum (II) complex in a therapeuticallyeffective amount, and a pharmaceutically acceptable carrier, whichcomposition is effective to inhibit sarcoma S 180 in baboons.